Protein synthesis is a fundamental biological process that underlies the development of polypeptide therapeutics, vaccines, diagnostics, and industrial enzymes. With the advent of recombinant DNA (rDNA) technology, it has become possible to harness the catalytic machinery of the cell to produce a desired protein. This can be achieved within the cellular environment or in vitro using lysates derived from cells.
In vitro, or cell-free, protein synthesis offers several advantages over conventional in vivo protein expression methods. Cell-free systems can direct most, if not all, of the metabolic resources of the cell towards the exclusive production of one protein. Moreover, the lack of a cell wall and membrane components in vitro is advantageous because it allows for control of the synthesis environment. For example, tRNA levels can be changed to reflect the codon usage of genes being expressed. The redox potential, pH, or ionic strength can also be altered with greater flexibility than with in vivo protein synthesis because concerns of cell growth or viability do not exist. Furthermore, direct recovery of purified, properly folded protein products can be easily achieved.
This invention relates to in vitro polypeptide synthesis in a system utilizing a suitable cell lysate. In particular, the invention provides a method for monitoring changes in the profile of various components (such as protein quantity and state of modification) within a cell-free in vitro polypeptide synthesis system, allowing the identification of components that correlate in their level or amount to the cell lysate's capacity to produce recombinant proteins in the system. The invention also provides a method for improving protein yield from the in vitro polypeptide synthesis reactions, by way of countering the changes in cell lysate components responsible for reduced protein yield, for example, by down-regulating proteins that have been identified to suppress the in vitro polypeptide synthesis and/or up-regulating proteins that have been identified to promote the in vitro polypeptide synthesis in the cell-free system. Because of the significant advantages of a cell-free in vitro polypeptide synthesis system due to its relative simplicity, there exists the need to improve the system's productivity and efficiency. The present invention fulfills this and other related needs.